X tremegene hp

For immunofluorescent labeling of TAP-tagged constructs, SK-N-AS cells were seeded out on glass coverslips in a 6-well plate at a density of 40 000 cells/cm² prior transfection. 24 h after seeding, the cells were transfected with 1 μg plasmid DNA using X-tremegene HP (Roche) for 48 h in a ratio of 1:3 (μg DNA:μl X-tremegene HP). After 48 h incubation, transfected cells were washed with 2x2mL of ice cold 1xPBS. Subsequently the cells were fixed and permeabilized with -20°C methanol for 5 min followed by washing with 3x2mL of ice cold 1xPBS. Cells were then blocked in 1x Tris-buffered saline (TBS, 137mM NaCl, 2,7mM KCl, 25mM Tris-Base, pH 7,4) containing 5% bovine serum albumin (BSA) and 1% Tween-20 at room temperature for 1 h. Thereafter, cells were incubated with an Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (Invitrogen, A11055) and DRAQ5 (for DNA staining) at concentrations 1:2000 and 1:5000 respectively, for 1 h at room temperature. Note, TAP-tagged Fe65 was directly detected by the anti-goat antibody due to the protein A domain. The cells were washed 3x500μL/coverslip in 1xTBS containing 1% Tween-20 before mounting on glass slides with Fluoromount-G (SouthernBiotech, 0100–01). Subcellular distribution of immunostained TAP-tagged Fe65 was determined using an imaging system with a Nipcov spinning disc (CSU-22) on a Zeiss Axiovert 200.

Fibroblast CHO-K1 cells, were from American Type Culture Collection(ATCC) and maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS). The cells, plated in 35-mm dishes and grown to confluence, were transfected with 2 μg of DNA and 4 μl of X-treme Gene HP (Roche, Indianapolis, IN), or co-transfeced with 1 μg of each DNA and 4 μl of X-treme Gene HP following the manufacturer's instructions. The cells were used 18-24 h post transfection.

(Day 0) HeLa-mCAT8 cells (1.5×10⁵ cells/well in 12-well plates) were cultured overnight. (Day 1) A pair of TALEN plasmids was mixed with X-tremeGENE HP (Roche Diagnostics) (in 12-well plates, 0.5 µg each of plasmids and 2 µl X-tremeGENE HP were mixed in 100 µl Opti-MEM), and then the mixture was added to the cells. (Day 2) The cells were transferred to 6-well plates and cultured at 37°C with puromycin at 5 µg/ml, which is higher than the usual concentration to concentrate cells with a higher expression of TALENs. (Day 3) The plates were moved to 30°C. (Day 4) The medium was changed to puromycin-free medium and the cells were kept at 30°C. (Day 7) The cells were subcultured and grown at 37°C for a few days. The TALEN-treated HeLa cells were harvested for indel analysis and lysenin treatment, or diluted to isolate gene-disrupted clones.

cDNAs coding for mouse Wnt3a and Wnt5a, monoclonal antibodies against mouse Wnt3a and Wnt5a, and L cell stably expressing mouse Wnt3a (L-3a) (Shibamoto et al., 1998 (link)) were all kindly provided by Shinji Takada (Okazaki Integrated Bioscience Center, Japan). The Open Source Wnt Project plasmids containing ORF clones for human Wnt proteins (Najdi et al., 2012 (link)) were obtained from Addgene. A cDNA coding for the human afamin was a generous gift from Luc Bélanger (Research Center, L’Hotel-Dieu de Québec, Canada). cDNAs coding for the following proteins were obtained by DNA synthesis based on their reported nucleotide sequences; bovine AFM (NM_001192175), human serum albumin (BC034023), human AFP (BC027881), and mouse VDBP (AK010965). A highly pure, carrier-free recombinant mouse Wnt3a protein was purchased from R&D Systems (catalogue No. 1324-WNP-010/CF). HEK293T, HEK293S GnT1 (kindly provided by G. Korhana), and L-3a cells are maintained in basal media containing DMEM (for HEK lines) or DMEM/F12_1:1 (for L-3a), supplemented with 10% fetal calf serum (FCS). All cell transfection experiments were performed using X-tremeGENE HP (Roche).